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1. Introduction
2. A brief history of RNA interference (RNAi)
3. Increased activity of the CS enzyme
4. Landmark paper
5. Timeline of important RNAi discoveries
6. Biochemical pathways of Dicer and RISC
7. Dicer substrate hypothesis
8. Design of dsRNA and nomenclature used in study
9. Longer duplexes can be more potent
10. Map of EGFP target sites
11. Improved potency at EGFP site-2 and site-3
12. Targeting endogenous genes hnRNPH and La
13. Dose response curves for EGFP-S1
14. Experiments "in vitro" with various dsRNAs
15. 27mer is cleaved "in vivo" to 21 mer size
16. Results of dicing EGFP-S1 27+0 "in vitro"
17. Tiling experiment
18. 27mers can have longer duration of silencing (1)
19. 27mers can have longer duration of silencing (2)
20. 27mers do not trigger IFN or PKR responses
21. 27mer Dicer-substrate dsRNAs as triggers of RNAi
22. Example: suppression of STAT1 gene
23. Not all 27mers work well - STAT-1 data
24. 27mers work better at a TNF-alfa target site
25. 27mers work better at a Stratifin target site
26. Why do some 27mers work better than others?
27. Effect of end modification on dicing
28. Understanding dicing patterns
29. EGFP-S3 blunt 27mer dicing pattern
30. The variety of blunt 27mers dicing patterns
31. SAR - structure activity relationship study (1)
32. Summary of SAR ESI - dicing studies
33. Mass Spec dicing results for EGFP-S1 L-form
34. Mass Spec dicing results for EGFP-S1 R-form
35. Two different 27mers can produce the same 21mer
36. R 27mer is more potent than L 27mer at EGFP-S1
37. R 27mer is more potent than L 27mer at EGFP-S2
38. R-form more potent for two sites of hnRNPH
39. R-form more potent for TNF
40. Effect of dicing on 27mer functional asymmetry
41. Prior work that may explain R vs. L potency
42. Influence of 27mer asymmetry on RISC loading
43. R-form favors "AS" strand loading into RISC
44. SAR - structure activity relationship study (2)
45. The potency of duplexes >30 bp
46. RNA is better than DNA in 3'-overhang
47. 3'-overhang length does not matter
48. Analysis of all possible 3'-overhang sequences
49. Central mutations disrupt function
50. Test of the potency of the new 27 mer duplexes
51. Results of testing the new 27 mer duplexes
52. Can new 27 mer designs be used "in vivo"?
53. Suppression of galectin-1 by 27mer dsRNA
54. Summary
55. Credits
56. Other collaborating scientists in the project
57. Dicer substrate RNAi publications
58. References (1)
59. References (2)
60. References ( 3)

Topics Covered

• Potency of RNA duplexes of ~27 nucleotides in length
• Dicer substrates
• Variation in dicing patterns
• Defined substrate preferences for Dicer
• Design of RNA duplexes that are processed to specific, desired products
• Dicer processing introduces functional asymmetry into the potency of a RNA duplex
• Preferential binding of Dicer to 3'-overhand
• The exploitation of Dicer properties to improve design of RNAi reagents

Links

Series:

• RNA Interference

Categories:

• Biochemistry
• Methods

Talk Citation

Publication History

• Published on October 1, 2007
• Archived on October 12, 2016

Financial Disclosures

• Dr. Mark Behlke has not informed HSTalks of any commercial/financial relationship that it is appropriate to disclose.